Ryanodine receptors (RyR2) regulate intracellular Ca2+ release from the sarcoplasmic reticulum (SR), and are important determinants of cardiac contractility. Chronic Ca2+/calmodulin-dependent protein kinase (CaMKII) phosphorylation at serine 2814 (S2814) on RyR2CaMKII has been recognized as a principal cause of enhanced SR Ca2+ leak, which contributes to contractile dysfunction in CHF. The long-term goal of this project is to define the cellular/molecular mechanisms by which CaMKII regulates RyR2-mediated SR Ca2+ release, cardiac contractility, and cardiac hypertrophy in normal, failing, and athletic hearts by studying knockin mice in which the CaMKII phosphorylation site serine 2814 (S2814) on RyR2 is either inactivated (S2814A) or constitutively activated (S2814D). Our overall hypothesis is that chronic CaMKII phosphorylation of RyR2 enhances diastolic leak of Ca2+ from the SR, which interferes with SR Ca2+ loading, causing initial hypertrophy and eventual depressed contractility of the heart. To test this hypothesis, we propose to: Aim 1: To determine whether genetic inhibition of CaMKIIS phosphorylation of RyR2 in mice ameliorates the development of pathological hypertrophy. In patients with heart failure, there are complex changes in Ca2+ homeostasis. Our hypothesis is that inhibition of CaMKII phosphorylation of S2814 on RyR2 inhibits aberrant SR Ca2+ release and pathological hypertrophy in S2814A knockin mice. Aim 2: Evaluate whether inhibition of CaMKII5 phosphorylation of RyR2 is the primary mechanism by which exercise training benefits mice with heart failure. In patients with sustained HP, exercise training can relieve symptoms of cardiac dysfunction, improve exercise capacity, and reduce mortality. The rationale of these experiments is to determine the role of CaMKII-mediated phosphorylation of RyR2 in the exercise- related improvement of heart failure patients. Methods include echocardiography, transverse aortic constriction (TAC), intracardiac hemodynamic measurements, cell isolation and confocal microscopy, MRI, exercise/treadmill training, immunohistochemistry, histology, and RT-PCR. Abnormal Ca2+ regulation in the heart muscle cell could lead to development of HP. In this project, we will use a genetic approach to test whether inhibition of Ca2+ leak leads to prevention of hypertrophy and heart failure development.